The object of the invention is a method for inducting targeted somatic transgenesis (TGC=targeted genetic conditioning), which is used for expressing foreign proteins in cells, tissue, organ or an entire host organism, as well as for somatic gene therapy.
It is known that proteins for technical application or for therapeutic purposes can be expressed in sufficient quantity by the transfer of genes in microorganisms or mammalian cells. These procedures are particularly important for proteins occurring naturally in the body, such as hormones, regulatory factors, enzymes, enzyme inhibitors and humanized monoclonal antibodies which are otherwise only available to a limited extent or not available at all. The procedures are also important for producing surface proteins of pathogenic microorganisms or viral envelope proteins so as to safely produce diagnostic tests and for the development of efficacious vaccines. Through protein engineering it is also possible to produce new types of proteins, which through fusion, mutation or deletion of the corresponding DNA sequences, have properties optimized for particular uses, for example immunotoxins.
Genes obtained from human cells are also functional in mouse, rat or sheep cells and there lead to the formation of corresponding gene products. This has already been made use of in the production of therapeutic products, for example in the milk of transgenic farm animals. The hitherto known method has been by the microinjection of corresponding foreign DNA carrying vectors into the nucleus of the fertilized egg cell, in which the DNA is then incorporated into the chromosome with a yield of 1%. The transgenic fertilized egg cell is then transplanted into hormonally stimulated mother animals. An offspring carrying the transfected gene in all its body cells is the basis for the creation of a xe2x80x9ctransgenic herd/flockxe2x80x9d. Using gene technology it is now possible to alter farm animals in such a targeted way that they produce human proteins in their blood, tissue or milk, which cannot be produced by microorganisms or plants.
However, the use of transgenic animals as protein production factories has the decisive disadvantage that it is necessary to manipulate the germ line of the animal. Due is to the considerable expenditure of technology and time required to create and breed transgenic animals and also due to the discussions regarding the ethical consequences of these methods, alternative methods for producing proteins in animal hosts without manipulation of the germ line are necessary and would be very advantageous.
It is known, furthermore, that the milk of mammals such as cows, sheep, goats, horses or pigs can contain a range of disease-causing bacterial agents. Among such agents are Listeria, mycobacteria, Brucella, Rhodococcus, Salmonella, Shigella, Escherichia, Aeromonads and Yersinia or general bacteria with intracellular lifestyle [1, 2]. These bacteria are usually transmitted to humans or animals through oral ingestion [3], but can also be transmitted by droplet infection. A major source for the infection of humans with Listeria [4], mycobacteria [5] and Escherichia coli is contaminated milk [6]. Humans ingest the bacteria when consuming unpasteurised milk or milk products. The other bacteria types listed above, such as Salmonella, Shigella, Yersinia, Rhodococcus and Brucella are transmitted to humans in a similar way. However, bacteria may also enter humans through other bacterially infected animal products from cows, goats, sheep, hares, horses, pigs or poultry.
The infection of animals frequently occurs through mucosal surfaces and very frequently through the digestive tract. However, after ingestion of bacteria, for example in the case of Listeria, not all tissues show symptoms of infection. In cows and goats the infection is mainly evident in the udder, spleen and liver. In sheep there may additionally be illness in the central nervous system in the form of meningitis, so not all animals survive the infection. With infection of the udder, the infection chain is closed. With contaminated milk, acquired bacteria can reinfect another animal, for example a suckling calf or a human via the digestive tract.
The following is known at present regarding the process of bacterial infection in humans, here presented using the example of Listeria:
Of the six known Listeria species, only L.monocytogenes and L.ivanoviiare pathogenic for humans [7]. Illness in humans results from consuming infected milk or milk products. The course of the illness depends on the state of health of the individual and is generally inapparent. Intrauterine transmission of bacteria to the fetus may occur during pregnancy, resulting in abortion, stillbirth or premature birth. In all cases excellent and problem-free treatment exists using antibiotics such as ampicillin or erythromycin [8; 8a].
The mode of entry into the cell occurs is well defined for L.monocytogenes in humans and animals and for L.ivanovii in sheep. For full pathogenicity of Listeria to occur, a range of pathogenicity factors are necessary. Among them are PrfA (positive regulator of virulence), ActA (actin nucleating protein), PlcA (phosphatidylinositol-specific phopholipase), PlcB (phosphatidylcholine-specific phopholipase), Hly (listeriolysin), Mpl (metalloprotease) [9]. The cell specificity of the pathogenxe2x80x94host cell interaction is mediated through a range of proteins. Among these are the internalins InlA and InlB, which are involved in the initial contact and the interaction of bacteria and cell surface [10, 11]. Under experimental conditions L.monocytogenes can also infect endothelial cells, epithelial cells, fibroblasts and hepatocytes. In addition, L.monocytogenes can infect cells of the white blood cell count like neutrophilic granulocytes, macrophages and lymphocytes. This is a significant factor in the transmission of bacteria from the site of primary infection to the target organ in the host. Finally, lung tissue can also be infected by Listeria if the bacteria are applied as a droplet infection.
After adhering to the cell surface, L.monocytogenes is taken up by the cell by endocytosis, the bacterium breaks down the endosome membrane under the effect of listeriolysin (Hly) and is thus released into the cell cytosol [14]. Once inside the cell, the bacteria can proliferate. With the production of further proteins, the fully pathogenic bacteria does not stay localized but actively spreads to distal sites . Bacterial spread is effected by using a range of proteins from L.monocytogenes itself and some cellular proteins [15, 16]. ActA is expressed on the cell surface of L.monocytogenes. It binds the cellular protein VASP, which for its part forms the bridge required for the attachment of cellular actin. Actin tails subsequently develop, which carry the bacterium at their tip and thus move it further through the cell. If L.monocytogenes contacts the cell membrane, a membrane protrusion forms, which projects directly into any adjacent cells if they are present. This protrusion is then endocytosed by the adjacent cell so the L.monocytogenes is then inside the new cell within a double membrane. The two membranes are dissolved under the effect of Hly and PlcB [171]. At the end of this process L.monocytogenes has also infected the neighbouring cell and the infection process begins again. In this way L.monocytogenes enters, for example, secretory cells of the cow udder. Secreted Listeria proteins are detectable in milk, i.e. they are passed on intracellularly from the lactating cell into the milk [18]. Hly (listeriolysin) and IrpA (internalin related protein [19]) are two pathogenicity factors belonging to this group of proteins which are produced, secreted and passed out in milk in large quantities by L.monocytogenes [20].
Knowledge of the infection process has made it possible to alter L.monocytogenes genetically in such a way that it expresses foreign proteins. Examples for the expression of foreign proteins in L.monocytogenes are: alkaline phosphatase from Escherichia coli, nucleoprotein from influenza virus, major capsid protein (L1) from cottontail rabbit papillomavirus (CRPV) and Gag protein from HIV type 1 [20 to 27].
In addition to proteins of prokaryotic origin, this also applies to viral proteins which are not normally produced within eukaryotic cells. These viral proteins and similar foreign proteins of prokaryotic and eukaryotic origin can be produced by L.monocytogenes without a eukaryotic cell being needed. Proteins produced by L.monocytogenes are secreted into the milk.
Infection by bacteria occurs through specific interactions of ligand proteins of the bacteria with receptor proteins of the target cells. In the case of L.monocytogenes, the internalin family plays a significant role; the internalin proteins determine to a large extent the cell specificity of the infection process [28]. Additionally, an ActA dependent cell ingestion has been discussed, which is mediated through receptors of the heparan sulphate family [29]. If L.monocytogenes infects a cell, it does not lead to a full infection cycle in every case. If listeriolysin in L.monocytogenes is inactivated, the bacteria then remain in the endosome and the infection in the xe2x80x9cfirst cellxe2x80x9d does not take place. Bacteria in which the protein ActA is deleted, inactive or no longer available, enter the first infected cell but remain there and can no longer infect the neighbouring cells [30, 31]. If PclB is deleted, the bacteria is no longer able to establish itself in the second cell.
L.monocytogenes is a bacterium which can be treated with a range of antibiotics. Ampicillin and penicillin (always in combination with gentamycin) are particularly suitable. Erythromycin and sulphonamides can also be used as alternatives. Tetracycline, vancomycin or chloramphenicol can also be used in special cases [32]. Similar treatments exist for other bacteria [8a] of the following types: Aeromonads, Bartonella, Brucella, Campylobacter, Enterobacteriaceae, Mycobacterium, Renibacterium, Rhodococcus and other bacteria which are genetically or biochemically related to them.
Given this information, the question arises as to how bacterial infection can be used to induce organotropic protein production.
This problem is solved by a TGC procedure that induces targeted somatic transgenesis, whereby bacteria, carrying a foreign DNA which is integrated into an episomal vector and prepared for subsequent transcription and expression, release their genetic information into an infected single cell when infecting cells, tissue, an organ or the whole host organism and so cause expression of the foreign protein.
This method can be used to obtain a foreign protein but is also advantageous for somatic gene therapy. Here the foreign DNA, introduced into the host organism through bacterial infection, can cause the production of protein missing in the host organism or, by producing single or double strand nucleic acids, can increase, reduce or hinder the production of a protein in the host organism. This method can be used on all known farm animals and also on humans.
If the infected tissue is the egg of a poultry bird, the foreign protein is produced in the egg and can be isolated following known procedures for the isolation of proteins, for example from hen eggs. If the infected tissue is blood cell tissue, the bacteria can spread via parenteral infection of the cells and through them the foreign DNA can reach the entire infected organism. If the host animals are laboratory animals whose infected organ is an udder, the desired foreign protein is then produced in the milk of the laboratory animal from which the foreign protein can then be isolated.